1. Field of the Invention
This invention relates in general to biological assays and diagnostic assays and, in particular, to such assays conducted on optical bio-discs. More specifically, but without restriction to the particular embodiments hereinafter described in accordance with the best mode of practice, this invention is further directed at sample preparation methods for use in cellular assays and related optical bio-disc systems.
The present invention may be advantageously employed in combination with any of the discs, assays, and systems disclosed in the following commonly assigned and co-pending patent applications: U.S. Provisional Application Ser. No. 60/302,757 entitled “Clinical Diagnostic Optical Bio-Disc And Related Methods For Selection And Detection Of Lymphocytes Including Helper-Inducer/Suppressor-Cytotoxic Cells” filed Jul. 3, 2001; U.S. Provisional Application Ser. No. 60/306,035 entitled “Quantitative and Qualitative Methods for Cell Isolation and Typing Including Immunophenotyping” filed Jul. 17, 2001; U.S. Provisional Application Ser. No. 60/305,993 entitled “Capture Layer Assemblies and Optical Bio-Discs for Immunophenotyping” filed Jul. 17, 2001; U.S. Provisional Application Ser. No. 60/306,592 entitled “Methods for Imaging Blood Cells, Blood-Borne Parasites and Pathogens, and Other Biological Matter Including Related Optical Bio-Discs and Drive Assemblies” filed Jul. 19, 2001; U.S. Provisional Application Ser. No. 60/307,263 entitled “Quantitative and Qualitative Methods for Cell Isolation and Typing Including Immunophenotyping” filed Jul. 23, 2001; U.S. patent application Ser. No. 10/233,322 entitled “Capture Layer Assemblies for Cellular Assays Including Related Optical Analysis Discs and Methods” filed Aug. 30, 2002; and U.S. patent application Ser. No. 10/236,857 entitled “Nuclear Morphology Based Identification and Quantification of White Blood Cell Types Using Optical Bio-Disc Systems” filed Sep. 6, 2002. All of these applications are herein in incorporated by reference.
2. Discussion of the Related Art
Blood cell counts are used during diagnosis, treatment, and follow-up to determine the health of the patient. Complete blood count (CBC) is a collection of tests including hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, and white blood cell count. Blood count is the enumeration of the red corpuscles and the leukocytes per cubic mm of whole blood.
White Blood Cell Count (WBC, leukocytes) is the total number of white blood cells in a standard sample of blood. In a normal healthy person, typically the WBC counts are 4000 to 10800 cells per microliter (μl). Factors such as exercise, stress, and disease can affect these values. A high WBC may indicate infection, leukemia, or tissue damage. There is an increased risk of infection when the WBC count falls below 1000 cells per microliter.
Leukocyte differential testing is essential to gather information beyond that obtainable from the leukocyte count itself. Leukocyte differential count is used to evaluate newly suspected infection or fever (even if the CBC is normal), suspicion of a disorder associated with abnormalities, an abnormal leukocyte count, suspected leukemia, and other abnormalities such as eosinophilia, monocytosis, or basophilia. Repeated testing for leukocyte or leukocyte differential may be performed in the presence of severe leukopenia (e.g., secondary to drug therapy). During treatment, for example during chemotherapy or radiation therapy, blood counts are very important to determine whether the treatment is depleting healthy blood cells in addition to cancerous cells. Since chemotherapy affects the production of blood cells, it is important to check the amount of various kinds of cells in the blood.
Differential leukocyte counts are determined by computerized cell counting equipment. Such apparatus determine the total count and the percentages of the five major white cell types. In normal individuals, there are a majority of neutrophils (50–60%), followed by lymphocytes (20–40%), then monocytes (2–9%), with a few eosinophils (1–4%) and basophils (0.5–2%).
Within the category of lymphocytes there are further sub-types of cells. For example, lymphocytes can be broadly divided into T-cells (thymus-derived lymphocytes) and B-cells (bursal-equivalent lymphocytes), which are largely responsible for cell-mediated and humoral immunity respectively. Although morphological characteristics have been used to classify groups within the leukocytes, morphology alone has proved inadequate in distinguishing the many functional capabilities of lymphocyte sub-types. To distinguish lymphocytes with various functions, techniques including analysis by rosetting, immuno-fluorescence microscopy, enzyme histochemistry, and recently, monoclonal antibodies against unique cell surface markers have been developed.
T-cells are often further distinguished by the presence of one of two major cell surface antigens such as CD4 and CD8. Type CD4+ cells are referred to as helper T-cells and are involved in antibody-mediated immunity. These T-cells bind to antigens presented by B-cells and cause the development of a clone of plasma cells which secrete antibodies against the antigenic material. The CD4+ T-cells are also essential for cell-mediated immunity. It is understood that CD4+ T-cells bind to antigen presented by antigen-presenting cells (APCs) such as phagocytic macrophages and dendritic cells, and release lymphokines that attract other immune system cells to the area. The result is inflammation, and the accumulation of cells and molecules that attempt to wall off and destroy the antigenic material.
Type CD8+ T-cells are referred to as cytotoxic or killer T-cells. These T-cells secrete molecules that destroy the cell to which they have bound. This is important in fighting viral infections, since the CD8+ T-cells destroy the infected cells before they can release a fresh crop of viruses that are able to infect other cells.
Human immunodefiency virus is a retrovirus with high affinity for CD4+ T cells and therefore CD4+ T cells are potent targets for the virus. Acquired immune deficiency syndrome (AIDS) provides a vivid and tragic illustration of the importance of CD4+ T cells in immunity. The human immunodeficiency virus (HIV) binds to CD4+ molecules and invades and infects CD4+ T cells. As the disease progresses, the number of CD4+ T cells declines below its normal range of about 1000 per microliter (ul). One of the explanations may be the unceasing effort of the patient's CD8+ T cells to destroy the infected CD4+ cells. Alternately, uninfected CD4+ cells may be induced to commit suicide (apoptosis).
When the number of CD4+ T cells drop below 400 per microliter, the ability of the patient to mount an immune response declines dramatically. Not only does the patient become hypersusceptible to infections from pathogens that invade the body including microorganisms, especially viruses and fungi that normally inhabit our tissues without harming us. Eventually the patient dies of opportunistic infections like Candidiasis, Cytomegalovirus, Herpes simplex viruses, Pneumocystis carinii, pneumonia, Toxoplasmosis, Tuberculosis and others.
In addition to CD4 and CD8, there are many other cell surface antigens (for example, CD3, CD16, CD19, CD45, and CD56) which can be used to identify sub-types of lymphocytes. The ability to detect these cell surface antigens by antibody techniques has added a new dimension to diagnostic pathology, and a variety of techniques are available for the study of immunophenotypes of hematolymphoid disorders (e.g., AIDS, leukemias, and lymphomas). Conventional microimmuno-assays such as radio-immunoassays (RIA), enzyme-immunoassay (EIA), fluorescence-immunoassay (FIA) use an isotope, an enzyme, or a fluorescent substance in order to detect the presence or absence of corresponding antibodies or antigens, respectively, that react specifically therewith.
The number of platelets in a standard sample of blood typically is 133,000 to 333,000 platelets per microliter (μl). An excess number of platelets is called thrombocythemia. Above normal platelet counts may be due to a reactive response or bone marrow failure. Reactive responses are typically caused by bleeding, infection, neoplasia, and myeloproliferative disorders. Bone marrow failure usually involves loss of blood cells known as pancytopenia. On the other hand, decreased platelet counts are due to immune thrombocytopenia. Thrombocytopenia occurs if the platelet count fall below 30,000, which results in abnormal bleeding. Counts below 5000 are considered life threatening.
A number of therapeutic approaches are used to treat AIDS including protease inhibitors, reverse transcriptase inhibitors, Integrase inhibitors and others. Estimation of CD4+ and CD8+ T lymphocyte numbers and their ratio (CD4+/CD8+ T-lymphocytes) are critical to assess immune health of human patients with immune-compromised diseases and to assess the effectiveness of treatment.
The ability to detect cell-associated antigens by antibody techniques has added a new dimension to diagnostic pathology. A variety of techniques are available for the study of immunophenotypes of hematolymphiod disorders. Conventional microimmunoassays like radio-immunoassays (RIA), enzyme-immunoassay (EIA), fluorescence-immunoassay (FIA) use an isotope, an enzyme or a fluorescent substance in order to detect the presence or absence of corresponding antibodies or antigens, respectively, that react specifically therewith. However the above methods have limitations and disadvantages. RIA requires special installations, precautions, limited half-life and various other factors. Methods using enzyme or fluorescence substances as labels is measured by determining coloring or luminescence require sensitive, sophisticated instruments to detect the calorimetric or fluorescent reactions in addition to requiring several washing steps to remove excess, unbound, un-reacted reagents. Furthermore, application of the above methods of detection for cells particularly lymphocytes and cancer cells and the like specimens, needs improvement in technology for the preparation, detection and analysis in high efficiency.
A tool developed around the use of fluorescent antibody specific for cell-surface antigens is the technique of fluorescence-activated cell sorting (FACS) of flow cytometry. This is a very reliable, fast and sensitive method. Flow cytometric analysis is performed on whole blood sample that is subjected to RBC lysis leaving the leukocytes intact. White cells of interest are then labeled with fluorescent markers for identification with the FACS scanner. The foremost requirement of a sample for flow cytometric analysis is that the sample is in a monodisperse suspension and desired cells are labeled with fluorescent markers. It is very high-priced test and the whole system requires handling by a trained technician in a clinical analysis laboratory and an expensive instrument. Another disadvantage with flow cytometry is that the cells once analyzed are no longer available for repeated analysis or additional investigation for example microscopic examinations of rare event cells.
Surface marker analysis is a very useful laboratory tool, which has been particularly very useful in studying leukemias, lymphomas and immunodeficiency diseases. Antibody-based micro-array technologies certainly are the state-of-the art technology, particularly in clinical diagnostics, for identification of specific antigens in a sample. Most diagnostic tests require determination of only a limited panel of analytes (such as in of cancers, leukemia, lymphoma, thyroid disease, etc.). Therefore, the requirements by a miniaturized technology for only a very small amount of blood sample and the savings in time and cost of laboratory personnel, upon simultaneous measurement of all the clinically relevant parameters in a single test are likely to prove compelling attractive to hospital laboratories and point-of-care facilities due to its cost-effectiveness, labor effective and its simplicity.
We have developed a simple, inexpensive system for analyzing specific cell surface antigens and performing data analysis in a fraction of time and cost compared to conventional methods. This system employs specially prepared optical bio-discs, related detection assemblies, and supporting software and processing methods.
Blood samples require processing prior to analysis similar to the protocol used for FACS scanner. Erythrocytes (red cells) due to their abundant numbers can interfere with specific binding of leukocytes (white cells). Therefore, prior to analyzing a sample with FACS scanner, blood samples are incubated with RBC lysing buffer that destroys red cells excluding the white cells.
In commonly assigned U.S. Provisional Application No. 60/308,197 filed Jul. 27, 2001, we describe a method developed and designed to perform helper/inducer-suppressor/cytotoxic assay on an optical bio-disc by isolating mononuclear cells from the whole blood using a density gradient protocol. In the present invention, we described a microfluidic chamber or circuit that can be used to perform the lysis procedure on the disc and the leukocyte material left over is dispelled into the assay or analysis chambers that have been layered with specific capture antibodies for helper/inducer-suppressor/cytotoxic assay. This particular chamber design may include two interconnected chambers. Chamber one contains the lysis buffer. Blood sample to be analyzed is injected into the “lysis” chamber. Incubated for 15 minutes for the lysis to be complete. Once the lysis is complete, the disc is spun to enable the remaining leukocytes to pass into the analysis chamber that has been coated with specific capture antibodies. Following incubation of chemistry with the cell sample for 30 minutes, the disc is read and imaged with the laser optics and the images analyzed with an appropriate software.